Project Summary Neurotransmitter release critically depends on the precise assembly of the secretory machine. Within a presynaptic nerve terminal, synaptic vesicles exclusively fuse at the active zone, a protein scaffold that forms release sites opposed to postsynaptic receptors. This scaffold consists of RIM, ELKS, Liprin-? and other active zone specific proteins. It also contains many proteins that are important for secretion and synaptic structure, but that are not restricted to the active zone. In the past two decades, research from many laboratories has started to provide deep insight into the functions of individual proteins at the active zone. However, much less is known about the assembly mechanisms of this key protein scaffold. This is particularly true for vertebrate synapses, perhaps because no genetic mutation to date has strongly disrupted the active zone scaffold. We here overcome this limitation by generating conditional knockout mice to simultaneously delete RIM and ELKS in hippocampal neurons. This mutation leads to massive disruption of the presynaptic active zone scaffold with loss of most of its vital components and of vesicle docking. Based on extensive preliminary data, we hypothesize that RIM and ELKS are redundantly required for release site assembly and function, and that these scaffolding proteins are recruited to the active zone by Liprin-?. We designed three specific aims to address our overarching hypothesis. In the first aim, we rigorously test synaptic structure and synaptic vesicle docking in these active zone disrupted neurons. We propose rescue experiments with individual proteins or protein domains to evaluate the molecular hierarchy of the recruitment of active zone proteins and to dissect mechanisms for vesicle docking. In aim 2, we use the mutants with disrupted active zones to test two models that are prominent in the field: we will determine whether the active zone and docking are required for synaptic vesicle release and we will test whether the active zone targets release to the membrane domain opposed to postsynaptic receptors. Our preliminary data reveal that fusion competent vesicles persist upon disruption of the active zone and loss of vesicle docking, which is surprising given the dogma that fusion competent vesicles are docked. In aim 3, we address molecular mechanisms of active zone assembly upstream of RIM and ELKS. The most parsimonious interpretation of the literature and our preliminary data is that Liprin-? recruits RIM and ELKS for active zone scaffolding. We systematically test this hypothesis in newly generated Liprin-? knockout mice. This is the first study that addresses vertebrate Liprin-? function using a rigorous genetic approach. This grant application will generate new knowledge on the mechanisms of vertebrate active zone assembly and function. Human genetic studies have identified mutations in many active zone proteins, including in RIM and in ELKS, which contribute to neurological disease. Thus, precise knowledge of active zone assembly is important for understanding synaptic function in health and disease.